Our proposal centers on the development of a novel antiviral compound for therapy of hepatitis B patients. Hepatitis B surface antigen (HBsAg) is an essential viral gene product, and its high levels in the circulation may play a role in immunosuppression. Current hepatitis B therapeutics are not directed at reducing this viral antigenemia, and are limited by either emergence of escape mutants, limited efficacy or toxicity. Phase I of our project is to test the feasibility of developing a therapeutic compound to selectively reduce HBsAg. Using a secretion assay and our "in-house" diverse, 80,000 different compound library, we have identified an aromatically-substituted tetrahydro-tetrazolo-(1,5-a)-pyrimidine (TTP), called HBF-0259, that selectively inhibits HBsAg secretion from tissue cultures with an EC50 of ~1.5 ?M. This is well below the concentration at which it is toxic, giving it a selectivity index of ~30. Unlike currently used HBV antiviral drugs, HBF-0259 is not an inhibitor of the polymerase, suggesting that it may be useful in cases where resistance has emerged. At 10 ?M, secretion of HBsAg, but not cellular glycoproteins was reduced by more than 80%, suggesting selectivity, and an excellent therapeutic opportunity. The Phase I proposal is to perform critical chemical and biological experiments to determine if this approach is practical and feasible. HBF-0259 has several chemical features that offer modification possibilities. Therefore, Phase I will: 1) Explore the chemistry and formulation of HBF- 0259 to develop an even better analogue; 2) test the active compounds against woodchuck hepatitis virus (WHV) for the design of animal studies, and against strains of HBV that are resistant to FDA-approved HBV antivirals in current clinical use; 3) determine the absorption, drug metabolism, extraction, and toxicity (ADMET) profiles of five active TTP analogues in the rat. Phase I studies will allow the selection of the two best compounds for advancement into Phase II , which will consist of testing for efficacy in the WHV model, and/or Phase I/II clinical trials in humans. A strength of this revised applications remains the well-conceived and straightforward experimental plan. The applicants addressed the major weakness, which was a lack of experimental details about chemical synthesis methods. The virological aspects of the proposed plan remain strong, and have been strengthen by a better plan to examine the emergence of resistant mutants. PUBLIC HEALTH RELEVANCE: This is a proposal for the development into a drug of a novel chemical family with activity against hepatitis B virus (HBV). This new drug would function through a different mechanism than the currently approved anti- hepatitis B drugs, and may be of use in multi-drug "cocktails" to minimize the emergence of resistant viruses, and to extend efficacy. At the end of the proposed project, the novel compound would be ready for large-scale Phase III clinical testing with hepatitis B patients. [unreadable] [unreadable] [unreadable]